Huntington's disease (HD) is caused by the expansion of a polyglutamine (polyQ) repeat in the huntingtin (htt) protein. The polyQ expansion in mutant htt causes the protein to misfold and aggregate in the neurons of HD patients, and this may contribute to pathogenesis. Why might the accumulation of aggregated htt in HD cause neurons to become dysfunctional and ultimately die? Our laboratory has hypothesized that small aggregated but soluble forms of htt called oligomers interact abnormally with other neuronal proteins in manner that impairs their ability to conduct their normal physiological functions. Recent studies also indicate that mutant htt is cleaved into different fragments, only some of which may contribute to HD pathogenesis. The major hypothesis that will be tested in this grant is that toxic mutant htt fragments, but not inert fragments, assemble into soluble oligomers that react with the molecular chaperones Hsp70/Hsp40, and that these oligomers correlate with neuronal dysfunction and toxicity. We will first determine if mutant htt fragments o different lengths that have been hypothesized to mediate pathogenesis in HD share the same propensity to form Hsp70/Hsp40-reactive oligomers. We will also determine if fragments hypothesized to be inert do not form such oligomers. Second, we will determine if these htt fragments form Hsp70/Hsp40-reactive oligomers in immortalized and primary neurons. We will also systematically compare the effects of these fragments on neuronal dysfunction and toxicity, and determine if dysfunction and toxicity correlate with levels of Hsp70/Hsp40-reactive oligomers. Finally, we will evaluate if Hsp70/Hsp40-reactive oligomers can be detected in brain homogenates from mouse models of HD. Together, these results will provide important information regarding the effects of amino acid context on mutant htt oligomer formation and its relationship to neuronal dysfunction and toxicity in vitro and in vivo. These results might also be informative for ongoing efforts to develop inhibitors of htt proteolysis and intrabodies as therapeutic approaches for HD. PUBLIC HEALTH RELEVANCE: In this study we will determine how amino acid context influences the ability of mutant huntingtin, the cause of Huntington's disease, to form soluble oligomeric aggregates that may contribute to pathogenesis in vitro and in vivo. The results from our studies will lead to a better understanding of the role of mutant huntingtin misfolding and aggregation in HD, and may help guide future therapeutic efforts aimed to remove toxic huntingtin species.